This application pertains to methods and compositions for non-invasive detection of complement-mediated inflammation using CR2-targeted nanoparticles.
Complement is the collective term for a series of blood proteins that constitute a major effector mechanism of the immune system. Complement plays an important role in the pathology of many autoimmune, inflammatory, and ischemic diseases. Inappropriate complement activation and its deposition on host cells can lead to complement-mediated cell lysis of target structures, as well as tissue destruction due to the generation of powerful mediators of inflammation.
Complement can be activated by one of the three pathways, the classical, lectin, and alternative pathways. The classical pathway is activated through the binding of the complement system protein C1q to antigen-antibody complexes, pentraxins, or apoptotic cells. The pentraxins include C-reactive protein and serum amyloid P component. The lectin pathway is initiated by binding of microbial saccharides to the mannose-binding lectin. The alternative pathway is activated on surfaces of pathogens that have neutral or positive charge characteristics and do not express or contain complement inhibitors. This results from the process termed “tickover” of C3 that occurs spontaneously, involving the interaction of conformationally altered C3 with factor B, and results in the fixation of active C3b on pathogens or other surfaces. The alternative pathway can also be initiated when certain antibodies block endogenous regulatory mechanisms, by IgA-containing immune complexes, or when expression of complement regulatory proteins is decreased. In addition, the alternative pathway is activated by a mechanism called the “amplification loop” when C3b that is deposited onto targets via the classical or lectin pathway then binds factor B. See e.g., H. J. Müller-Eberhard, 1988, Ann. Rev. Biochem. 57:321. For example, Holers and colleagues have shown that the alternative pathway is amplified at sites of local injury when inflammatory cells are recruited following initial complement activation. See e.g., Girardi et al., 2003, J. Clin. Invest. 112:1644. Dramatic complement amplification through the alternative pathway then occurs through a mechanism that involves either the additional generation of injured cells that fix complement, local synthesis of alternative pathway components, or more likely because infiltrating inflammatory cells that carry preformed C3, and properdin greatly increase activation specifically at that site.
Alternative pathway activation is initiated when circulating factor B binds to activated C3. This complex is then cleaved by circulating factor D to yield an enzymatically active fragment, C3bBb. C3bBb cleaves C3 generating C3b, which drives inflammation and also further amplifies the activation process, generating a positive feedback loop. Factor H is a key regulator (inhibitor) of the alternative complement pathway that competes with factor B for binding to C3b. Binding of C3b to Factor H also leads to degradation of C3b by factor Ito the inactive form C3bi (also designated iC3b), thus exerting a further check on complement activation. Factor H regulates complement in the fluid phase, circulating at a plasma concentration of approximately 500 μg/ml, but its binding to cells is a regulated phenomenon enhanced by the presence of a negatively charged surface as well as fixed C3b, iC3b, or C3d. See e.g., Jozsi et al., 2004, Histopathol. 19:251-258.
Complement activation and complement-mediated inflammation are involved in the etiology and progression of numerous diseases. The down-regulation of complement activation has been shown to be effective in treating several diseases in animal models and in ex vivo studies, including, for example, systemic lupus erythematosus and glomerulonephritis (Y. Wang et al., 1996, Proc. Nat'l Acad. Sci. USA 93:8563-8568), rheumatoid arthritis (Y. Wang et al., 1995, Proc. Nat'l Acad. Sci. USA 92:8955-8959), cardiopulmonary bypass and hemodialysis (C. S. Rinder, 1995, J. Clin. Invest. 96:1564-1572), hyperacute rejection in organ transplantation (T. J. Kroshus et al., 1995, Transplantation 60:1194-1202), myocardial infarction (J. W. Homeister et al., 1993, J. Immunol. 150:1055-1064; H. F. Weisman et al., 1990, Science 249:146-151), ischemia/reperfusion injury (E. A. Amsterdam et al., 1995, Am. J. Physiol. 268:H448-H457), antibody-mediated allograft rejection, for example, in the kidneys (J. B. Colvin, 2007, J. Am. Soc. Nephrol. 18(4):1046-56), and adult respiratory distress syndrome (R. Rabinovici et al., 1992, J. Immunol. 149:1744-1750). Moreover, other inflammatory conditions and autoimmune/immune complex diseases are also closely associated with complement activation (B. P. Morgan. 1994, Eur. J. Clin. Invest. 24:219-228), including, but not limited to, thermal injury, severe asthma, anaphylactic shock, bowel inflammation, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, myocarditis, membranoproliferative glomerulonephritis, atypical hemolytic uremic syndrome, Sjögren's syndrome, renal and pulmonary ischemia/reperfusion, and other organ-specific inflammatory disorders.
A variety of disorders are associated with inflammation, however, so definitive diagnosis of complement-mediated inflammation typically requires confirmation via immuno-staining or other in vitro analysis performed on tissue samples retrieved by biopsy. While biopsies are in many respects routine, they have their limitations and are not risk-free. Because commonly used needle or punch biopsies sample only a small portion of the target organ, there is a risk of sample error leading to an incorrect diagnosis. Furthermore, although biopsy is a generally safe procedure, major complications such as internal bleeding may occur in a significant number of cases.
In some cases, because of the difficulties in diagnosing disease or monitoring disease progression, for example, in patients with systemic lupus erythematosus or lupus nephritis, repeat renal biopsies are therefore frequently necessary to assess the response to therapy or to diagnose disease relapse. See e.g., S. Bajaj et al., 2000, J. Rheumatol. 27:2822-2826. Although renal biopsy is generally a safe procedure, complications may occur in 6% or more of biopsies and intra-renal bleeding and hematuria are common. Patients requiring repeat biopsies are at concomitantly greater risk of complications. See e.g., W. L. Whittier et al., 2004, J. Am. Soc. Nephrol. 15:142-147; D. C. Mendelssohn et al., 1995, Am. J. Kidney Dis. 26:580-585. Thus, a non-invasive method of detecting or accurately assessing the presence, degree and/or extent of complement-mediated inflammation would be of significant value in diagnosing disease, formulating treatment strategies and monitoring their efficacy for many inflammatory diseases, including lupus nephritis.
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